'Myosin V belongs to the myosin family of actin-based ATPase motor proteins involved in cellular movements with the actin cytoskeleton in eukaryotic cells. Myosin V functions in organelle, vesicle, and mRNA transport and has been implicated in normal vertebrate neuronal function. Interestingly, single molecules of myosin V containing two ATPase catalytic domains (or heads) move processively along actin filaments. Although the kinetic mechanism of non-processive, recombinant, single-headed myosin V has been characterized, the processes determining double-headed processive motility remain unclear. The overall goal of this proposal is to define the kinetic mechanism that accounts for the double-headed myosin V ATPase cycle in order to test several published models of myosin V processivity. The first aim is to determine if the heads of doubleheaded myosin V coordinate ATPase cycles during processive movement using steady-state measurements of ATPase activity. The second aim is to use transient kinetic methods to measure the rate and equilibrium constants defining the double-headed myosin V ATPase cycle.